Abstract Mycobacterium tuberculosis (Mtb) is a human pathogen that causes significant morbidity and mortality worldwide. An increasing number of tuberculosis cases are caused by multi-drug and extreme-drug resistant Mtb strains, underscoring the need for novel therapeutics. Mtb manipulates infected cells in order to achieve virulence and to this end Mtb needs to secrete proteins, which will interact with the host cell. The first type 7 secretion system (T7SS) was originally identified in mycobacteria and since then four additional systems have been described (ESX-1 through ESX-5). A core, four-gene sequence of the ESX-5 system has been duplicated three times in the Mtb genome (ESX-5a, ESX-5b and ESX-5c). Here we propose the novel hypothesis that the duplicated ESX-5 regions have an accessory function in the secretion of specific subgroups of ESX-5-secreted proteins. In AIM1 we plan to generate Mtb deletion mutants in order to determine the impact of the deletion of the ESX-5 accessory systems on host cell responses and virulence. In AIM2 we will use proteomics approaches to characterize the secretomes associate with the ESX-5 accessory systems. These studies will lay the groundwork to enable the identification of specific, ESX-5-secreted, effector proteins critical for Mtb virulence. The proposed research will thus ultimately aid in the design of better recombinant vaccines strains and/or the generation novel targets for drug development.